A last-minute goal from the substitute secured all three points for the home side as it beat Guus Hiddink's men.Vaclav Pilar's stoppage-time strike ensured the Czech Republic consigned Netherlands to a shock 2-1 Euro 2016 qualifying defeat in Prague.
Substitute Pilar was on hand to apply a simple finish one minute into added time in the Group A opener at Generali Arena to punish a dreadful mistake from Daryl Janmaat.
Newcastle United defender Janmaat attempted to cushion a header back to goalkeeper Jasper Cillessen but hit the post, and midfielder Pilar could not believe his luck as he turned the ball in from only a few yards out.
Lazio defender Stefan de Vrij looked to have ensured Guus Hiddink avoided defeat in his first competitive game since returning for a second spell as coach of his country when he headed home 10 minutes into the second half until the late twist.
Borek Dockal had put the home side in front with a stunning strike, and coach Pavel Vrba was able to mark his maiden competitive match in charge of the Czech Republic by securing his first win as an international coach courtesy of Pilar's last-gasp goal.
Netherlands failed to recreate the sort of performances that saw it finish third in the World Cup in Brazil and was made to pay.
Midfield duo Lukas Vacha and Dockal came into the Czech side along with striker David Lafata, while Hiddink opted to replace Jeremain Lens and Dirk Kuyt with in-form forward Memphis Depay and defender Joel Veltman.
Hiddink bemoaned his side's "naive" defending in its 2-0 defeat against Italy on Thursday and his team was at it again early on in Prague.
It needed an important intervention from De Vrij to block Vladimir Darida's shot after the visitor was caught napping when the Czechs took a short corner.
Netherlands failed to heed the warning and was behind 21 minutes in courtesy of a sublime finish from Dockal.
The Sparta Prague man beat Cillessen with a left-footed finish that went in off the post after Lafata had laid the ball off to him outside the penalty area.
Georginio Wijnaldum spurned a chance to equalize only five minutes later when he burst into the box unmarked, but his control let him down.
Hiddink made a tactical change seven minutes before halftime, with Luciano Narsingh replacing Veltman and Netherlands switched from three center backs to a back four.
The lively Depay came into the game brimming with confidence, having scored eight goals already this season for PSV, and he almost added an international goal to that tally when his long-range drive was tipped over by Petr Cech.
Narsingh showed a lack of composure when he surged into the area just before the break as Netherlands started to get into their stride.
The Czech Republic was pegged back 10 minutes into the second half, as De Vrij rose highest to head home Daley Blind's inviting cross from the left.
Robin van Persie's eyes lit up when he was presented with a sight of goal but would have been relieved to see the flag up for offside after Cech kept out his attempt on goal.
Pilar then had the final say as he converted from point-blank range after Janmaat got his header all wrong right at the death and was left red-faced.